Figure 4.
Prohibitin 2 is JI051 target. A, target identification by microsequencing analysis. Silver staining of proteins isolated by streptavidin beads upon incubation with a biotinylated version of JI051 (JITV14) before or after displacement with a 10-fold excess of JI130. DMSO and JIN05 were used as negative controls. Bands indicated by arrows were analyzed by microsequencing. B, validation of PHB2 as a target for JI051 by pulldown with a photoreactive probe. FLAG–PHB2-expressing HEK293 cells were incubated with NeutrAvidin beads together with JIN04 (negative control) or JITV10 (diazirine photoreactive moiety), with or without a 2.5-fold excess of JI051, prior to Western blotting with anti-FLAG antibody. Input represents the supernatant fraction of cell lysates. C, KD determination of JITV10. Human recombinant GST-tagged PHB2 protein was incubated with JITV10 before UV exposure and Western blotting with HRP-conjugated streptavidin or PHB2 antibody. The densitometry analysis of HRP–streptavidin signal was normalized to PHB2.